1. Field of the Invention
The present invention relates to a method and composition of matter for using quantitative measurements of fluorescence intensity to measure multiple subpopulations of particles from a single sample of particles by flow cytometric techniques.
2. Description of Related Art
Flow cytometry is a rapid, high precision technique for analysis and sorting of many different particles, including formed elements of blood and other biologic tissue cells. Using flow cytometry, particles can be counted and sorted by passing a fluid stream containing the particles through a light beam produced by a laser light source. The particles passing through the light beam scatter the illuminating light; measuring the intensity of scattered light at different angles provides information about the size, shape, density, and surface morphology of the particles. Fluorochrome-labelling of the particles to be analyzed provides an often used alternative to relying on differential refraction of light to analyze the particles. When fluorochrome-labelled particles are counted or sorted, the presence or absence of fluorescence within a selected wavelength range emitted by the labelled particles following excitation by the illuminating light is the parameter measured in making the analysis. Fluorochrome labelling has advantages especially when counting particles of biological origin, because, in comparison to methods relying on measuring light refraction, quantitation of specific biochemicals is possible.
For a great many applications, subset analysis, defined as distinguishing multiple subpopulations of particles in a single sample of particles, would afford great savings in time and expense. Commonly available flow cytometers, which include only one laser and two fluorescence detection channels, used in conjunction with conventional methods, however, are limited to measurement of not more than two fluorescent dyes, and thus, can distinguish no more than two subpopulations of particles in any one sample. Most efforts to enhance the number of subpopulations that can be distinguished in a single sample have relied on using highly sophisticated instruments. Such instruments contain two or more excitation lasers and a sufficient number of fluorescence detection channels to detect fluorescence from three or more fluorochromes. Even using these sophisticated instruments, the number of subpopulations which can be distinguished in a single sample is limited by the finite number of available fluorochromes. Additionally, widespread use of these sophisticated instruments, particularly for routine clinical diagnosis, is restricted by their prohibitively high cost.
Evidence that the need for a method of subset analysis using widely available instruments remains unfulfilled is provided by continuing efforts to develop such a method. In U.S. Pat. No. 4,499,052 to Fulwyler, a method of distinguishing multiple subpopulations of cells from a single sample of cells is described. This method employs several cell-specific antibodies having one hundred percent of the antibody molecules labelled with different, preselected ratios of fluorescein and rhodamine. After reaction with a reagent containing the labelled antibodies, the cells are distinguished and counted by comparing the measured fluorochrome ratios to the preselected fluorochrome ratios and summing the number of cells having each fluorochrome ratio.
Another method for using widely available instruments and fluorochrome-labelled antibodies for subset analysis that permits analysis of a limited number of subpopulations from a single sample recently has been described. Shapiro, H. M., Practical Flow Cytometry, 127-128 (1985). According to this method, a sample containing several different cell types is mixed with a reagent containing three different antibodies having each antibody molecule labelled with one fluorochrome. Antibodies specific to one cell type are labelled with fluorochrome A, antibodies specific to a second cell type are labelled with fluorochrome B, and antibodies specific to a third cell type are labelled with the fluorochromes A and B such that approximately one-half the third cell type-specific antibody molecules are labelled with fluorochrome A and the remaining third cell type-specific antibodies are labelled with fluorochrome B. All of the third cell type-specific antibodies have the same antigenic affinity, and thus the maximal measured intensity of each fluorochrome on the third cell type is less than the maximal measured intensity when antibodies having the same antigen affinity conjugated to one fluorochrome are used alone. After reaction with the reagent containing fluorochromes A and B, the subsets, upon passing through the excitation laser, emit light of different colors. For example, if fluorochrome A is red and fluorochrome B is green, the first cell type will emit only red light, the second only green light, and the third will emit red and green light. Thus, the three cell types are counted and separated by segregating red from green from red and green.
The procedures described in the above references have in common the use of fluorochrome-labelled antibodies having one hundred percent of the antibody molecules labelled with fluorochrome. Since precision dictates that the cells to be counted be labelled under antibody excess, cell separation has been restricted to qualitative distinctions between fluorochrome-labelled cells, that is, a cell either does or does not emit a certain color or either does or does not emit a ratio of colors equivalent to a preselected ratio of colors. Absent from the above references is a method of distinguishing subsets based upon quantitative measurements of fluorescence intensity.